Diabetes, also referred to as hyperglycemia, is a metabolic abnormality mainly relating to glucose metabolism, which is caused by, for example, insufficient secretion of insulin or decreased insulin sensitivity of the target cells, and is characterized by high blood sugar. When high blood sugar is sustained for a long period, serious complications in various organs and nerves such as retinopathy, nephropathy and neuropathy may occur mostly due to angiopathy. Therefore, it is currently very important in treatment of diabetes to control and keep blood sugar with the normal range, and means for controlling a blood sugar levels has been conventionally studied.
A glucose tolerance test (an oral load of 75 g glucose) is used for diagnosis of diabetes (hyperglycemia). For this diagnosis, blood is drawn during fasting to measure values of insulin and blood sugar in the blood, and blood is drawn again after a certain time period from intake of water having 75 g of glucose dissolved therein to measure values of insulin and blood sugar in the blood. When a value (ΔIRI/ΔBG) of “a difference in values of blood insulin 30 minutes after the load and before the load” (ΔIRI) divided by “a difference in sugar blood values 30 minutes after the load and before the load” (ΔBG) is not more than 0.4, it is determined that there is a high risk of serious exacerbation of hyperglycemia.
Diabetes is mainly classified into type 1 diabetes and type 2 diabetes. Type 1 diabetes is developed with absolute deficit of insulin secretion caused by debility or death of pancreatic β cells which leads to no or very low insulin secretion. This may be caused by virus infection and autoimmune abnormality originating from virus infection. Type 2 diabetes is developed when insulin secretion from pancreatic β cells is decreased (quantitative insufficiency of insulin), or when insulin action in glucose-uptake cells is decreased (insulin resistance) and, as insulin resistance increases, insulin becomes relatively quantitatively insufficient and blood sugar levels starts to increase. In the latter case, insulin secretion from pancreatic β cells becomes excessive to supply relative quantitative insufficiency of insulin, and when the excessive secretion of insulin reaches a maximum level and is maintained for a long time, pancreatic β cells are finally exhausted and insulin secretion from the cells is decreased.
Therefore, it is assumed that a fundamental cause of diabetes (hyperglycemia) is deficit of pancreatic β cells and decrease in production or insufficiency of secretion of insulin in the cells.
Though an insulin formulation, biguanide agents, sulfonylurea agents, thiazolidinedione agents and the like are currently used for improvement of blood sugar levels, hypoglycemic agents currently used are not yet satisfactory in terms of side effects and the like. Furthermore, since these agents are developed for the purpose of decreasing blood sugar levels, the agents can be used for symptomatic treatment to control blood sugar levels, but are not yet satisfactory from a viewpoint of a curative treatment to improve the fundamental cause of diabetes as described above, that is, deficit of pancreatic β cells themselves and decrease in production or insufficiency of secretion of insulin in the cells.
From the viewpoint as described above, development of an agent capable of promoting neogenesis or regeneration of pancreatic β cells, suppressing exhaustion or death of pancreatic β cells, and further, promoting insulin production in pancreatic β cells is eagerly desired for suppression or treatment of hyperglycemia.
Ghrelin, on the other hand, is a hormone discovered from rat stomach in 1999, which is a peptide having quite a unique chemical structure wherein the 3rd N-terminal amino acid is acylated by a fatty acid (Nature, 402, pp. 656-660, 1999). Ghrelin has been shown to have a function of stimulating secretion of growth hormone from pituitary gland, and has also been shown in a recent study to have a function such as stimulating food intake, or accumulating fat to increase body weight and improve cardiac function (Nature, 409, pp. 194-198, 2001; Endocr. Rev., 25, pp. 426-457, 2004; Front Neuroendocrinol., 25, pp. 27-68, 2004),
Ghrelin was isolated and purified from rat as an endogenous growth hormone secretagogue (GHS) for a growth hormone secretagogue receptor (GHS-R). Amino acid sequences of ghrelin having similar primary structures are also known in vertebrates other than rat, for example, human, mouse, porcine, chicken, eel, bovine, equine, ovine, frog, trout, and canine.
Human:GSS(n-octanoyl)FLSPEHQRVQQRKESKKPPAKLQPR(Sequence No. 1)GSS(n-octanoyl)FLSPEHQRVQRKESKKPPAKLQPR(Sequence No. 2) Rat:GSS(n-octanoyl)FLSPEHQKAQQRKESKKPPAKLQPR(Sequence No. 3)GSS(n-octanoyl)FLSPEHQKAQRKESKKPPAKLQPR(Sequence No. 4) Mouse:GSS(n-octanoyl)FLSPEHQKAQQRKESKKPPAKLQPR(Sequence No. 5) Porcine:GSS(n-octanoyl)FLSPEHQKVQQRKESKKPAAKLKPR(Sequence No. 6) Bovine:GSS(n-octanoyl)FLSPEHQKLQRKEAKKPSGRLKPR(Sequence No. 7) Ovine:GSS(n-octanoyl)FLSPEHQKLQRKEPKKPSGRLKPR(Sequence No. 8) Canine:GSS(n-octanoyl)FLSPEHQKLQQRKESKKPPAKLQPR(Sequence No. 9) Eel:GSS(n-octanoyl)FLSPSQRPQGKDKKPPRV-NH2(Sequence No. 10) Trout:GSS(n-octanoyl)FLSPSQKPQVRQGKGKPPRV-NH2(Sequence No. 11)GSS(n-octanoyl)FLSPSQKPQGKGKPPRV-NH2(Sequence No. 12) Chicken:GSS(n-octanoyl)FLSPTYKNIQQQKGTRKPTAR(Sequence No. 13)GSS(n-octanoyl)FLSPTYKNIQQQKDTRKPTAR(Sequence No. 14)GSS(n-octanoyl)FLSPTYKNIQQQKDTRKPTARLH(Sequence No. 15) Bullfrog:GLT(n-octanoyl)FLSPADMQKIAERQSQNKLRHGNM(Sequence No. 16)GLT(n-decanoyl)FLSPADMQKIAERQSQNKLRHGNM(Sequence No. 16)GLT(n-octanoyl)FLSPADMQKIAERQSQNKLRHGNMN(Sequence No. 17) Tilapia:GSS(n-octanoyl)FLSPSQKPQNKVKSSRI-NH2(Sequence No. 18) Catfish:GSS(n-octanoyl)FLSPTQKPQNRGDRKPPRV-NH2(Sequence No. 19)GSS(n-octanoyl)FLSPTQKPQNRGDRKPPRVG(Sequence No. 20) Equine:GSS(n-butanoyl)FLSPEHHKVQHRKESKKPPAKLKPR(Sequence No. 21)(In a notation above, an amino acid residue is represented by one letter.)
The above-described peptide has a specific structure wherein a serine residue (S) or a threonine residue (T) in the 3rd position has a side chain hydroxyl group acylated by a fatty acid such as octanoic acid or decanoic acid. Except for ghrelin, there is no example of biologically active peptide having such a hydrophobic modified structure isolated from an organism. This peptide is known to have a potent growth hormone releasing activity and participate in regulation of growth hormone secretion (International Publication WO01/07475).
Since ghrelin and a receptor thereof (GHS-R) are also expressed in a pancreas (Endocrinology, 145, pp. 3813-3820, 2004; Brain Res. Mol. Brain Res., 48, pp. 23-29, 1997; J. Clin. Endocrinol. Metab., 87, pp. 1300-1308, 2002), studies have been made relating to glucose metabolism or insulin secretion, which showed that ghrelin regulates insulin and glucose in blood, and functions of ghrelin to increase blood sugar and suppress or promote insulin secretion have been reported (Pancreas, 27, pp. 161-166, 2003; Endocrinology, 144, pp. 916-921, 2003; Eur. J. Endocrinol., 146, pp. 241-244, 2002; Endocrinology, 143, pp. 185-190, 2002; J. Neuroendcrinol., 14, pp. 555-560, 2002). A function of ghrelin for pancreatic β cells to promote neogenesis or regeneration of the cell, however, has not been suggested.
In addition, though International Publication WO2001/56592 (and US Patent Publications 2001/0020012A1 and 2004/0063636A1) suggests use of a GHS-R 1A receptor ligand including ghrelin as a pharmaceutical for treatment of type 2 diabetes, there is no demonstration as to whether ghrelin can be used as a remedy for type 2 diabetes or not, and whether utility as suggested actually exists or not is not known to those skilled in the art. In addition, though International Publication WO2002/60472 describes a function of ghrelin for obesity, utility for treatment of diabetes has not been demonstrated.
As for the relationship between pancreatic β cells and ghrelin, a phenomenon has been recognized wherein increased ghrelin producing cells (ε cells) substitute for pancreatic β cells when differentiation to β cells is suppressed (Proc. Natl. Acad. Sci., 101, pp. 2924-2929, 2004). Though a possibility of the two kinds of cells being derived from an identical precursor cell, and a future possibility of producing pure β cells group from stem cells or the like using ghrelin producing cells to treat diabetes at a cell level have been suggested, these are mere possibilities and have not been substantiated. In addition, this publication does not suggest or teach that ghrelin has a function of promoting neogenesis or regeneration of pancreatic β cells or promotes insulin production in pancreatic β cells.